[Crystallization of a covalently-linked complex of adrenodoxin and adrenodoxin reductase]. / Kristallizatsiia kovalentno-sshitogo kompleksa adrenodoksina i adrenodoksinreduktazy.

Two forms of mitochondrial adrenodoxin reductase from bovine adrenals and recombinant bovine adrenodoxin and adrenodoxin reductase expressed in Escherichia coli were isolated, purified to homogeneity and biochemically characterized. Recombinant adrenodoxin reductase was expressed as a single polypeptide; its retention time on DEAE-Fractogel coincides with the second form (F2) of the mitochondrial reductase. Two enzyme forms have similar adrenodoxin reductase activities in two types of systems comprising either cytochrome c or cytochrome P-450 (11 beta) as the terminal electron acceptor. Adrenodoxin and each of two reductase forms were cross-linked using 1-ethyl-3-(dimethyl-amino-propyl)carbodiimide. An effective two-step method for the purification of the active heterologous cross-linked complexes is suggested that enables purification of the functional complexes to homogeneity. The cross-linked bimolecular complex of adrenodoxin and adrenodoxin reductase was crystallized for the first time.

[Chemical modification of steroid-hydroxylating monooxygenases with fluorescein isothiocyanate]. / Khimicheskaia modifikatsiia steroidgidroksiliruiushchikh monooksigenaz fluorestseinizotiotsianatom.

Chemical modifications of cytochrome P-450scc and cytochrome P-450(11) beta with fluorescein-, diiodofluorescein-, eosine- and rhodamine isothiocyanate have been carried out. At a low reagent/protein ratio and neutral pH, a selective chemical modification was known to take place which did not affect the spectral properties of cytochrome P-450scc. Covalent chromatography was found useful to discriminate between covalent modification of cytochrome P-450scc and non-specific binding of FITC with cytochrome P-450scc. Proteolytic modification of cytochrome P-450scc and structural analysis indicate that a lysine residue of the C-terminal sequence of cytochrome P-450scc is accessible to FITC. The residue was shown, by the analysis of the chymotryptic hydrolysate of the fragment F2, to be Lys338. Effect of modification with FITC on the interaction of cytochrome P-450scc with cholesterol or adrenodoxin, on the reduction kinetics and on the conversion of cholesterol to pregnenolone was also studied.

[The study of the active site of cytochrome P-450 LM2 using the chemical modification of tyrosine residues by tetranitromethane]. / Issledovanie aktivnogo tsentra tsitokhroma P-450 LM2 s pomoshch'iu khimicheskoi modifikatsii ostatkov tirozina tetranitrometanom.

Selective chemical modification of the hemoprotein by tetranitromethane was used in order to elucidate the functional role of tyrosine residues in the cytochrome P-450 LM2 molecule. It was shown that the degree of cytochrome P-450 LM2 modification can be determined, using the second derivative of the UV absorption spectra. Modification of one tyrosine residue resulted in the inactivation of cytochrome P-450 LM2. Nitration of the cytochrome was accompanied by changes in the spectral properties of the hemoprotein with the formation of spectra typical of hyperporphyrin structures, thus suggesting the involvement of tyrosine residues in the formation of the active center of cytochrome P-450 LM2.

[Absorption and magnetic circular dichroism spectra of hemoproteins in nonequilibrium states. V. Cytochrome P450 and its substrate complex]. / Issledovanie spektrov pogloshcheniia i magnitnogo krugovogo dikhroizma gemoproteinov v konformatsionno neravnovesnykh sostoianiiakh. V. Tsitokhrom P450 i ego kompleksy s substratami.

It was shown that ferrocytochrome P450 forms a nonequilibrium state if ferrocytochrome P450 and its complexes are reduced in freezed water-glycerol solutions by thermolysed electrons, arising during gamma-radiolysis of the matrix at 77 degrees K. Unlike the equilibrium form of ferrocytochrome P450 with the heme iron at the high-spin state the reduced nonequilibrium form of the protein contains the heme iron at a low-spin state. The absorption spectrum of ferrocytochrome P450 in the nonequilibrium state is characterized by alpha and beta-bands at 562 and 534 nm, respectively, whereas the magnetic circular dichroism spectra exhibit type A effect at 562 nm. Upon temperature increasing the nonequilibrium state is relaxed to the equilibrium one. Type 1 substrates had practically no influence on the spectral characteristic of the nonequilibrium form of ferrocytochrome P450. Binding of type 2 substrates results in an essential decrease of the intensity ratio of the alpha- and beta-bands (A alpha/A beta) and is accompanied by a red-shift of the alpha-band and corresponding magnetic circular dichroism effect. It was shown that mercaptoethanol complex of hemoglobin, formed by reduction at 77 degrees K is spectrally similar to the nonequilibrium ferrocytochrome P450 complex with type 2 substrates. From analysis of experimental data one can conclude that (i) the ligand environment of heme iron in oxidased and reduced cytochrome P450 are different; (ii) the sixth axial ligand of the heme iron in the oxidised protein is probably a water molecule (OH-) attached by a hydrogen bond to the neighbouring histidine. It is assumed that a similar nonequilibrium form of cytochrome P450 can be formed in physiological conditions.